ANALYZING THE RED-SHIFT CHARACTERISTICS OF AZULENIC,NAPHTHYL, OTHER RING-FUSED AND RETINYL PIGMENT ANALOGS OF BACTERIORHODOPSIN*

Prompted by the near infrared-absorbing properties of some of the azulenic bacteriorhodopsin (bR) analogs, we have analyzed their absorption characteristics along with 11 new related ring-fused analogs and the corresponding Schiff bases (SB) and protonated Schiff bases (PSB). The following three factors are believed to contribute to the total red shift of each of the pigment analogs (α_RS): perturbation of the basic chromophore (SB shift, ΔSB), protonation of the SB (PSB shift, PSBS) and protein perturbation (the opsin shift, OS). For each factor, effects of structural modifications were examined. For the red-shifted pigments, percent OS has been suggested as an alternate way of measuring protein perturbation. Computer-simulated chromophores provided evidence against any explanation involving altered shapes of the binding pocket as a major cause for absorption differences. Implications of the current bR results on preparation of further red-shifted bR and possible application to visual pigment analogs are discussed.     

Quantum yield of CHAPSO-solubilized rhodopsin and 3-hydroxy retinal containing bovine opsin.

The quantum yields of bleaching for two artificial pigments, bovine opsin combined with (3R)-3-hydroxy retinal or (3R,S)-3-methoxy retinal, were determined in comparison to the value for regenerated bovine rhodopsin. Regeneration of the visual pigments was performed by incubation of 3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate (CHAPSO)-solubilized opsin with the 11-cis isomers of retinal and the respective retinal derivatives. The extinction coefficients of the pigments in CHAPSO were determined to 35,000 M-1 cm-1 (native rhodopsin), 35,300 M-1 cm-1 (regenerated rhodopsin) and 34,500 M-1 cm-1 (3-OH retinal opsin). With respect to rhodopsin (lambda max: 500 nm), the pigments carrying the substituted chromophores exhibit blue shifted absorbance maxima (3-hydroxy and 3-methoxy retinal opsin: 488 nm). In parallel experiments under absolutely identical conditions we find related to the value of CHAPSO solubilized rhodopsin (identical to 1) a quantum efficiency of bleaching for the 3-hydroxy pigment of 1.2.     

A semiempirical study of the optimized ground and excited state potential energy surfaces of retinal and its protonated Schiff base.

The electronic ground and first excited states of retinal and its Schiff base are optimized for the first time using the semiempirical AM1 Hamiltonian. The barrier for rotation about the C(11)-C(12) double bond is characterized by variation of both the twist angle delta(C(10)-C(11)-C(12)-C(13)) and the bond length d(C(11)-C(12)). The potential energy surface is obtained by varying these two parameters. The calculated ground state rotational barrier is equal to 15.6 kcal/mol for retinal and 20.5 kcal/mol for its Schiff base. The all-trans conformation is more stable by 3.7 kcal/mol than the 11-cis geometry. For the first excited state, S(1,) the 90 degrees twisted geometry represents a saddle point for retinal with the rotational barrier of 14.6 kcal/mol. In contrast, this conformation is an energy minimum for the Schiff base. It can be easily reached at room temperature from the planar minima since it is separated from them by a barrier of only 0.6 kcal/mol. The 90 degrees minimum conformation is more stable than the all-trans by 8.6 kcal/mol. We are thus able to present a reaction path on the S(1) surface of the retinal Schiff base with an almost barrier-less geometrical relaxation into a twisted minimum geometry, as observed experimentally. The character of the ground and first excited singlet states underscores the need for the inclusion of double excitations in the calculations.     

Rhodopsin regeneration is accelerated via noncovalent 11-cis retinal-opsin complex--a role of retinal binding pocket of opsin

The regeneration of bovine rhodopsin from its apoprotein opsin and the prosthetic group 11-cis retinal involves the formation of a retinylidene Schiff base with the epsilon-amino group of the active lysine residue of opsin. The pH dependence of a Schiff base formation in solution follows a typical bell-shaped profile because of the pH dependence of the formation and the following dehydration of a 1-aminoethanol intermediate. Unexpectedly, however, we find that the formation of rhodopsin from 11-cis retinal and opsin does not depend on pH over a wide pH range. These results are interpreted by the Matsumoto and Yoshizawa (Nature 258 [1975] 523) model of rhodopsin regeneration in which the 11-cis retinal chromophore binds first to opsin through the beta-ionone ring, followed by the slow formation of the retinylidene Schiff base in a restricted space. We find the second-order rate constant of the rhodopsin formation is 6100+/-300 mol(-1) s(-1) at 25 degrees C over the pH range 5-10. The second-order rate constant is much greater than that of a model Schiff base in solution by a factor of more than 10(7). A previous report by Pajares and Rando (J Biol Chem 264 [1989] 6804) suggests that the lysyl epsilon-NH(2) group of opsin is protonated when the beta-ionone ring binding site is unoccupied. The acceleration of the Schiff base formation in rhodopsin is explained by stabilization of the deprotonated form of the lysyl epsilon-NH(2) group which might be induced when the beta-ionone ring binding site is occupied through the noncovalent binding of 11-cis retinal to opsin at the initial stage of rhodopsin regeneration, followed by the proximity and orientation effect rendered by the formation of noncovalent 11-cis retinal-opsin complex.     

THE CONFORMATIONAL ANALYSIS AND PHOTOISOMERIZATION OF RETINOCHROME ANALOGS WITH POLYENALS

Abstract— 3, 7-Dimethyl-2, 4, 6, 8, 10-dodecapentaenal was synthesized for reconstitution of the retinochrome analog. Its opsin shift was 1000 cm ¹ smaller than that of native retinochrome, whose chromophore contains the same number of double bonds. The conformational change from 6-s-trans to 6-s-cis , as figured in a retinal molecule, plays an important role in the formation of the retinochrome analog, based on the estimation of opsin shifts for retinal analogs locked in the 6-s conformation. Thus the conformation of the 6–7 single bond in the native retinochrome was suggested to be 6 -cis . Analysis of the circular dichroic spectra of retinochrome analogs revealed that the 6-s conformation is independent of the appearance of the β-band. The stereoselectivity in the photoisomerization of the retinal analogs by a retinochrome template depends on the hydrophobic binding in the region of the β-ionone ring.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

Recent bioorganic studies on rhodopsin and visual transduction

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.     

Reaction path analysis of the "tunable" photoisomerization selectivity of free and locked retinal chromophores

Multiconfigurational second-order perturbation theory computations and reaction path mapping for the retinal protonated Schiff base models all-trans-nona-2,4,6,8-tetraeniminium and 2-cis-nona-2,4,6,8-tetraeniminium cation demonstrate that, in isolated conditions, retinal chromophores exhibit at least three competing excited-state double bond isomerization paths. These paths are associated with the photoisomerization of the double bonds in positions 9, 11, and 13, respectively, and are controlled by barriers that favor the position 11. The computations provide a basis for the understanding of the observed excited-state lifetime in both naturally occurring and synthetic chromophores in solution and, tentatively, in the protein environment. In particular, we provide a rationalization of the excited-state lifetimes observed for a group of locked retinal chromophores which suggests that photoisomerization in bacteriorhodopsin is the result of simultaneous specific "catalysis" (all-trans --> 13-cis path) accompanied by specific "inhibition" (all-trans --> 11-cis path). The nature of the S(1) --> S(0) decay channel associated with the three paths has also been investigated at the CASSCF level of theory. It is shown that the energy surfaces in the vicinity of the conical intersection for the photoisomerization about the central double bond of retinal (position 11) and the two corresponding lateral double bonds (positions 9 and 13) are structurally different.     

An opsin shift in rhodopsin: retinal S0-S1 excitation in protein, in solution, and in the gas phase

We considered a series of model systems for treating the photoabsorption of the 11-cis retinal chromophore in the protonated Schiff-base form in vacuum, solutions, and the protein environment. A high computational level, including the quantum mechanical-molecular mechanical (QM/MM) approach for solution and protein was utilized in simulations. The S0-S1 excitation energies in quantum subsystems were evaluated by means of an augmented version of the multiconfigurational quasidegenerate perturbation theory (aug-MCQDPT2) with the ground-state geometry parameters optimized in the density functional theory PBE0/cc-pVDZ approximation. The computed positions of absorption bands lambdamax, 599(g), 448(s), and 515(p) nm for the gas phase, solution, and protein, respectively, are in excellent agreement with the corresponding experimental data, 610(g), 445(s), and 500(p) nm. Such consistency provides a support for the formulated qualitative conclusions on the role of the chromophore geometry, environmental electrostatic field, and the counterion in different media. An essentially nonplanar geometry conformation of the chromophore group in the region of the C14-C15 bond was obtained for the protein, in particular, owing to the presence of the neighboring charged amino acid residue Glu181. Nonplanarity of the C14-C15 bond region along with the influence of the negatively charged counterions Glu181 and Glu113 are found to be important to reproduce the spectroscopic features of retinal chromophore inside the Rh cavity. Furthermore, the protein field is responsible for the largest bond-order decrease at the C11-C12 double bond upon excitation, which may be the reason for the 11-cis photoisomerization specificity.     

EFFECT OF PROTONATION ON THE ISOMERIZATION PROPERTIES OF n-BUTYLAMINE SCHIFF BASE OF ISOMERIC RETINAL AS REVEALED BY DIRECT HPLC ANALYSES: SELECTION OF ISOMERIZATION PATHWAYS BY RETINAL PROTEINS

Alumina adsorption chromatography and ion-pair reversed-phase chromatography were developed to analyze the isomers of unprotonated and protonated n-butylamine Schiff base of retinal (RSB and PRSB), respectively. Photoisomerization starting from the all-trans, 11-cis and 13-cis isomers was traced for RSB in n-hexane, acetonitrile, methanol and 1-butanol, and for PRSB in methanol, acetonitrile and 1-butanol. The quantum yields of photoisomerization for the all-trans, 9-cis, 11-cis and 13-cis isomers were determined for RSB and PRSB in the above solvents except 1-butanol. On the other hand, photoisomerization of isomeric retinal bound (through Schiff base linkage) to bovine serum albumin (RBSA) in aqueous solution (pH 3, 7 and 12) as well as thermal isomerization of RSB (in n-hexane), PRSB (in methanol) and RBSA (in aqueous solution, pH 7) were traced starting from the all-trans, 11-cis, and 13-cis isomers. Protonation of RSB drastically changes the pathway of photoisomerization and increases the quantum yields of isomeric RSB. The solvent polarity increases the quantum yields of RSB differently depending on the configuration. Protonation enhances thermal isomerization also. The results of the above model systems are compared with those of retinal proteins to rationalize their selection of the particular isomerization pathways.     

Torsion potential works in rhodopsin

We investigate the role of protein environment of rhodopsin and the intramolecular interaction of the chromophore in the cis-trans photoisomerization of rhodopsin by means of a newly developed theoretical method. We theoretically produce modified rhodopsins in which a force field of arbitrarily chosen part of the chromophore or the binding pocket of rhodopsin is altered. We compare the equilibrium conformation of the chromophore and the energy stored in the chromophore of modified rhodopsins with those of native rhodopsins. This method is called site-specific force field switch (SFS). We show that this method is most successfully applied to the torsion potential of rhodopsin. Namely, by reducing the twisting force constant of the C11=C12 of 11-cis retinal chromophore of rhodopsin to zero, we found that the equilibrium value of the twisting angle of the C11=C12 bond is twisted in the negative direction down to about -80 degrees. The relaxation energy obtained by this change amounts to an order of 10 kcal/mol. In the case that the twisting force constant of the other double bond is reduced to zero, no such large twisting of the bond happens. From these results we conclude that a certain torsion potential is applied specifically to the C11=C12 bond of the chromophore in the ground state of rhodopsin. This torsion potential facilitates the bond-specific cis-trans photoisomerization of rhodopsin. This kind of the mechanism is consistent with our torsion model proposed by us more than a quarter of century ago. The origin of the torsion potential is analyzed in detail on the basis of the chromophore structure and protein conformation, by applying the SFS method extensively.     

Asymmetric toggling of a natural photoswitch: ultrafast spectroscopy of Anabaena sensory rhodopsin

Photochemistry in retinal proteins (RPs) is determined both by the properties of the retinal chromophore and by its interactions with the surrounding protein. The initial retinal configuration, and the isomerization coordinates active in any specific protein, must be important factors influencing the course of photochemistry. This is illustrated by the vast differences between the photoisomerization dynamics in visual pigments which start 11-cis and end all-trans, and those observed in microbial ion pumps and sensory rhodopsins which start all-trans and end in a 13-cis configuration. However, isolating these factors is difficult since most RPs accommodate only one active stable ground-state configuration. Anabaena sensory rhodopsin, allegedly functioning in cyanobacteria as a wavelength sensor, exists in two stable photoswitchable forms, containing all-trans and 13-cis retinal isomers, at a wavelength-dependent ratio. Using femtosecond spectroscopy, and aided by extraction of coherent vibrational signatures, we show that cis-to-trans photoisomerization, as in visual pigments, is ballistic and over in a fraction of a picosecond, while the reverse is nearly 10 times slower and kinetically reminiscent of other microbial rhodopsins. This provides a new test case for appreciating medium effects on primary events in RPs.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

The role of the beta-ionone ring in the photochemical reaction of rhodopsin

Time-dependent density functional theory (TDDFT) calculations on the photoabsorption process of the 11-cis retinal protonated Schiff base (PSB) chromophore show that the Franck-Condon relaxation of the first excited state of the chromophore involves a torsional twist motion of the beta-ionone ring relative to the conjugated retinyl chain. For the ground state, the beta-ionone ring and the retinyl chain of the free retinal PSB chromophore form a -40 degrees dihedral angle as compared to -94 degrees for the first excited state. The double bonds of the retinal are shorter for the fully optimized structure of the excited state than for the ground state suggesting a higher cis-trans isomerization barrier for the excited state than for the ground state. According to the present TDDFT calculations, the excitation of the retinal PSB chromophore does not primarily lead to a reaction along the cis-trans torsional coordinate at the C11-C12 bond. The activation of the isomerization center seems to occur at a later stage of the photo reaction. The results obtained at the TDDFT level are supported by second-order M?ller-Plesset (MP2) and approximate singles and doubles-coupled cluster (CC2) calculations on retinal chromophore models; the MP2 and CC2 calculations yield for them qualitatively the same ground state and excited-state structures as obtained in the density functional theory and TDDFT calculations.     

THE ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

7,8-dihydro retinals outperform the native retinals in conferring photosensitivity to visual opsin

The visual pigment rhodopsin presents an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta II of this G protein-coupled receptor. The responsible ligand, retinal, is covalently bound to Lys-296 of the protein in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 5,6-dihydro- or 7,8-dihydro analogues might facilitate photoisomerization of a 9-Z and an 11-Z bond. Here we describe pigment analogues generated with bovine opsin and 11-Z 7,8-dihydro retinal or 9-Z 7,8-dihydro retinal. Both isomers readily generate photosensitive pigments that differ remarkably in spectral properties from the native pigments. In addition, in spite of the more flexible 7,8 single bond, both analogue pigments exhibit strikingly efficient photoisomerization while largely maintaining the activity toward the G-protein. These results bear upon the activation of ligand-gated signal transducers such as G protein-coupled receptors.     

Spectral sensitivity, structure and activation of eukaryotic rhodopsins: activation spectroscopy of rhodopsin analogs in Chlamydomonas.

Retinal normally binds opsin forming the chromophore of the visual pigment, rhodopsin. In this investigation synthetic analogs were bound by the opsin of living cells of the alga Chlamydomonas reinhardtii; the effect was assayed by phototaxis to give an activation spectrum for each rhodopsin analog. The results show the influence of different chromophores and the protein on the absorption of light. The maxima of the phototaxis action spectra shifted systematically with the number of double bonds conjugated with the imine (C = N+H) bond of the chromophore. Chromophores lacking a beta-ionone ring, methyl groups and all C = C double bonds photoactivated the rhodopsin of Chlamydomonas with normal efficiency. On the basis of a simple model involving one-electron transitions between occupied and virtual molecular orbitals, we estimate the charge distribution along the chromophore in the binding site. With this restraint we define a unique structural model for eukaryotic rhodopsins and explain the spectral clustering of pigments, the spectral differences between red and green rhodopsins and the molecular basis of color blindness. Our results are consistent with the triggering of the activation of rhodopsin by the light-mediated change in electric dipole moment rather than the steric cis-trans isomerization of the chromophore.     

Rhodopsin deactivation is affected by mutations of Tyrl91

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyrl91 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyrl91, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyrl91 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photo-isomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.     

Time-resolved resonance Raman analysis of chromophore structural changes in the formation and decay of rhodopsin's BSI intermediate

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.     

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.